During the previous funding period, we established an Nkx2.8-null mouse, in which the gene was replaced by a LacZ reporter gene under control of the intact Nkx2.8 promoter. In large airways of adult mice, reporter gene expression was confined to a subset of basal cells, and the number of expressing cells was significantly expanded in the Nkx2.8-/- mouse compared to Nkx2.8 heterozygotes. This cell expansion led to wide- spread bronchial dysplasia and aged mice had a high incidence of lung cancer. In development, Nkx2.8-expressing cells appeared relatively late, after the lungs had formed. First apparent in the distal lung buds, the cells spread upward throughout the bronchi and trachea, and eventually formed separate cell populations in the tracheo- bronchial and bronchioloalveolar regions. Both populations show properties of local stem cells. However, the null mutation deregulates only the tracheobronchial population. Increased LacZ-positive cells were associated with bronchial hyperplasia from birth, and labeling studies demonstrated that increased proliferation persisted in adults. Nkx2.8 therefore appears to act as a negative regulator that limits the number of tracheobron- chial stem cells. We propose new studies to accelerate the development of spontaneous bronchial cancer (Aim 1). The experiments will test the role of genetic background and suppressor-gene mutations on bronchial tumor progression. Other studies (Aim 2) will exploit the marker gene to analyze the dynamics of stem cell maintenance and differentiation, and their perturbation by the null-mutation. Histopathology and flow cytometry will correlate proliferation and differentiation markers in LacZ-expressing cells, which will also be isolated to characterize expression profiles, and differentiation properties in primary cell culture. Aim 3 will analyze tumor suppressive properties of induced Nkx2.8 expression in human lung cancer cells. Expression profiles from these cells will be com- pared to those obtained from mouse bronchial cell fractions, to determine specific relationships between tumor and stem cell regulation.